Ubiquitin (Ub), a 76 amino acid protein, is implicated in protein degradation and a myriad of cell signaling pathways, including DNA damage response, protein trafficking, cell-cycle progression, inflammation, immune response and regulation of apoptosis. Ubiquitylation occurs through formation of an isopeptide bond between the C-terminus of Ub and the ?-amino group of a lysine (Lys) residue on target proteins. Ub itself has seven Lys residues (K6, K11, K27, K29, K33, K48, and K63), each of which can be further ubiquitylated, generating poly-Ub chains ranging from 2 to 7 Ubs in length. The ability of Ub to form polymers through various lysines appears to be central to the versatility of this system in regulating cell processes. Research in the ubiquitin field is hampered by a lack of antibodies or other tools that selectively recognize poly-ubiquitylated proteins. To address this issue we have combined our experience of generating isopeptide selective poly-Ubs and yeast genetics to generate diverse single chain antibodies that will advance the field of cell biology. I this grant we propose to use yeast display of single domain camelid antibodies to select for linkage specific binders. We will employ synthetic peptides which encompass both the isopeptide bond and the sequence surrounding the lysine attachment site in ubiquitin as well as a complete set of di-ubiquitins comprising each of the 7 isopeptide linkages. Both synthetic peptide antigens and diUbs will be used individually to pan for antibodies. LifeSensors and the scientific community will use these tools to chart new pathways in cell biology and biochemistry. These tools will be used as detection agents in protein chemistry, clinical diagnostic applications and imaging studies. These tools will unmask ubiquitomics of the cellular proteins and uncover new roles of unique poly-ubiquitylated proteins. Given the poor nature of tools available for K48- and K63- poly-Ub linkages, as a proof-of- principle, the phase I grant will focus on K48- and K63-linkages and expand to the remaining linkages in Phase II. We believe that development of linkage selective tools will have as great an impact on cell biology as the development of phospho-specific antibodies had on cellular phosphorylation.